Abstract
Serine racemase (SR) is a brain enzyme present in glial cells, where it isomerizes L-serine into D-serine that, in turn, diffuses and coactivates the N-methyl-D-aspartate receptor through the binding to the so-called "glycine site." We have developed a method for the slow expression of SR in a eukaryotic vector that permits the correct insertion of the prosthetic group into the active site, rendering functional SR with a K(m) toward L-serine of 4.8 mm. Divalent cations such as calcium or manganese were necessary for complete enzyme activity, whereas the presence of chelators such as EDTA completely inhibited the enzyme. Moreover, direct binding of calcium to SR was evidenced using (45)Ca(2+). Gel filtration of the recombinant SR revealed the protein to be in a dimer-tetramer equilibrium. The addition of EDTA to a calcium-saturated serine racemase evokes a profound conformational change, as monitored by both fluorescence and circular dichroism techniques. Fluorescence titration allowed us to calculate a binding constant for calcium of 6.2 microm. Reagents that react with sulfhydryl groups, such as cystamine, were potent inhibitors of SR, in a clear reflection that one or more cysteine residues are important for enzyme activity. Additionally, 16 serine analogues were tested as a putative SR substrate or inhibitors. Significant inhibition was only observed for L-Ser-O-sulfate, L-cycloserine, and L-cysteine. Finally, activation of brain SR as a result of the changes in calcium concentration was studied in primary astrocytes. Treatment of astrocytes with the calcium ionophore, as well as with compounds that augment the intracellular calcium levels such as glutamate or kainate led to an increase in the amount of d-serine present in the extracellular medium. These results suggest that there might be a glutamatergic-mediated regulation of SR activity by intracellular calcium concentration.
Highlights
A novel enzyme responsible for the synthesis of D-serine in the brain and identified it as a 37-kDa pyridoxal phosphate-containing racemase present in astrocytes
Activation of Serine Racemase within Primary Astrocytes— we investigated whether the addition of glutamate, kainate, and the calcium ionophore A23187 to astrocytes could result in the intracellular activation of glial serine racemase, resulting in the extracellular secretion of D-Ser
Evidence for the role of astrocyte-derived D-serine as a neurotransmitter includes (i) its release following glutamatergic activation of non-NMDA receptors [7], (ii) the depression observed in NMDA transmission following selective degradation of D-serine by exogenous D-amino acid oxidase [15], (iii) the ability of D-serine to fully mimic the activity of the endogenous transmitter at the glycine site of the receptor [10, 13,14,15], (iv) the specific localization of the synthesized D-serine in synapses in close proximity to the NMDA receptors [7], and (v) the selective localization of serine racemase in astrocytes [5,6,7]
Summary
A novel enzyme responsible for the synthesis of D-serine in the brain and identified it as a 37-kDa pyridoxal phosphate-containing racemase present in astrocytes. These protoplasmic astrocytes typically ensheath synapses, strongly suggesting a role for D-serine in synaptic transmission. Selective degradation of extracellular D-serine using exogenously added amino acid oxidase greatly attenuates NMDA receptor-mediated neurotransmission, as monitored by the increase in neuronal nitric-oxide synthase activity or cGMP levels [15]. We demonstrate that both glutamate- and A23187 ionophore-induced calcium entrance in astrocytes lead to the activation of the serine racemase with a concomitant increase in the detected extracellular concentration of D-Ser
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